Transcript CyO / cn bw let-a?

Reading for Monday’s lecture: (genetic mosaics & chimeras)
p518 (“Aneuploid Mosaics…”)
pp731-732 (“What cells…”)
I will hold office hours during spring break, but at a different
time than normal:
during class time (11a-12N) on March 26 & March 30
Balancer chromosomes:
Using them to follow chromosomes in mutant screens
Consider the brute-force screen that led to the last fly Nobel Prize
N-V & W:
Aim: find genes that allow cells to know where they are
so the cells can know what they should be
expected lof mutant phenotype for “pattern formation” genes:
(genes generating
positional information)
(1) embryonic recessive lethal
vvvvvvvvvv
vvvvvvvvv
vvvvvvvv
vvvvvvv
vvvvvvvvvv
wildtype
polarity>>>
vvvvvvvvv
Post.
vvvvvvvv
Ant.
vvvvvvv
vvvvvvv
vvvvvvv
vvvvvvvvv
vvvvvvvvv
vvvvvvvvvv
vvvvvvvvvv
vvvvvvvvvv
vvvvvvvvv
vvvvvvvv
vvvvvvv
(2) alterred dentical belt pattern (exoskeleton)
in dead embryos (dying fly embryos can still differentiate a lot)
Post.
Post.
“bicaudal”
Second chromosome (brute force) screen
Second
dominant
secondtemperature- chromosome
sensitive
balancer
lethal
DTS / CyO
females
mutagenize
X
cn bw
males
@non-permissive
DTS / CyO
females
CyO / cn bw &
single sons
second-chromosome
“markers”
(eye color = white)
each son potentially
carries a new mutant
allele of interest
…but a different new mutant
in each
let??
take individual males
and mate separately
(10,000 crosses)
DTS / CyO
females
@non-permissive
CyO / cn bw let-a?
daughters
X
CyO / cn bw & let??
single sons
X
CyO / cn bw let-a?
sons
each group of progeny separately (forced incest):
unwanted sibs all die
CyO / CyO
DTS / CyO
DTS/ cn bw let?
CyO / cn bw let-a?
daughters
cn bw let-a?
X CyO /sons
each group of progeny separately (forced incest):
CyO / cn bw let-a?
to maintain any
new let mutation
cn bw let-a? / cn bw let-a?
CyO / CyO
do they all die? (no white eyes?)
and if so, when? how?
always die
only after a 2nd generation of 10,000 crosses
did they know which individual sons of mutagenized males
carried a recessive lethal mutation of interest (value)
Second chromosome screen
mutagenize
DTS / CyO
females
Brute force
cn bw
X
males
each son potentially
carries a new mutant
allele of interest
DTS / CyO
females
X
CyO / cn bw & mut??
F1 generation
single sons
CyO / cn bw mut-a?
daughters
cn bw mut-a?
X CyO / sons
cn bw mut-a? / cn bw mut-a?
F2 generation
keep populations
separate!
only after a 2nd generation of 10,000 crosses
did they know which original F1 sons carried mutations of value
…and if looking for maternal-effect mutations, go blindly one generation more!
Second chromosome screen
Temperature for
first cross doesn’t
really matter:
mutagenize
DTS / CyO
females
X
@non-permissive
OR permissive
DTS / CyO
females
X
cn bw
males
(1) have to handpick males anyway
CyO / cn bw & mut??
single
sons
or
(2) males have no
meiotic recombination
(so DTS/mut OK)
DTS / cn bw & mut??
@non-permissive
CyO / cn bw mut-a?
daughters
cn bw mut-a?
X CyO / sons
CyO / cn bw mut-a?
cn bw mut-a? / cn bw mut-a?
CyO / CyO
Classic N-V&W screen illustrates two important points:
(1) recessiveness (~lof) generally demands multiple generations
of blind forced incest crosses (mating siblings) to recover mutant
…can we overcome the limitations of recessiveness?
(2) recognizing an informative phenotype is a large part of the genetics game
The N-V & W advantage: an informative phenotype that could be scored
in dead embryos (didn’t demand survival -- or much else!). &Early
What if want to study something like eye development instead?
Attractive features: interesting AND non-essential (and more), but consider:
ey1 :recessive hypomorph, adults w/ no eyes
ey is pleiotropic
-(null)
ey
: recessive embryonic lethal
(multiple “unrelated”
phenotypes/functions)
Got lucky with ey1
how many other important eye genes missed?
…can we overcome the limitations of pleiotropy?
…can we overcome the limitations of pleiotropy?
YES…we shall overcome
but first:
already mentioned one way to deal with pleiotropy
temperature-conditional
mutant alleles
FAR
BETTER
ts muts. way
too limited
even
in
flies
& worms
(1) genetically sensitize the system:
turn lof recessives into dominants (but only with respect to
one non-essential aspect of the genes’ function)
(2) use targetted genetic mosaics to screen for recessives
in the F1 (homozygous clones in heterozygotes
…in non-essential tissues only!)
(1) genetically sensitize the system:
turn lof recessives into dominants (but only with respect to
one non-essential aspect of the genes’ function)
goal: make genes “artificially” haploinsufficient
Illustrate with example from fly eye development studies:
A developmental decision:
Stemmed from original observation:
R7 precursor cell
signal from
?
photo- R8 neighbor
cone photorecptr
cell recptr
sevenless/+ (wildtype)
vs. sev/sev R7 photoreceptor
missing
(turned into cone cell)
null sev allele: same thing
(hence, eye-specific)
bride-of-sevenless (null eye-specific)
null alleles not eye-specific:
pleiotropic: son-of-sevenless
seven-in-absentia
How many other pleiotropic genes missed?
seven-up
Other genes discovered
to be involved in the
R7 precursor decision:
(1) genetically sensitize the system:
turn lof recessives into dominants (but only with respect to
one non-essential aspect of the genes’ function)
make genes “artificially” haploinsufficient
R7 precursor cell
signal from
?
photo- R8 neighbor
cone photorecptr
cell recptr
How many pleiotropic genes missed?
R7 photoreceptor
missing
sev/sev
(turned into cone cell)
sev encodes v-src homolog
(human oncogene)
(3rd chromosome balancer)
sev- /sev- ; TM3,P{sevB4(ts)}/+
designer ts allele
growth
temperature
phenotype
screen for dominant mutations
that make:
24.3oC
R7 absent
R7 present (Dominant suppressors)
22.7oC
R7 present
R7 absent (Dominant enhancers)
(1) genetically sensitize the system:
turn lof recessives into dominants (but only with respect to
one non-essential aspect of the genes’ function)
growth
temperature
phenotype
screen for dominant mutations
that make:
24.3oC
R7 absent
R7 present (Dominant suppressors)
22.7oC
R7 present
R7 absent (Dominant enhancers)
Found many pleiotropic lof alleles of both types (incl. recessive lethals).
Poising sev+ activity level on a phenotypic threshold made other
genes haploinsufficient but only with respect to sev function!
Wildtype fly must normally have an excess of sev+ activity as insurance,
so it can tolerate fluctuations in levels of other genes in pathway during development
…if take away that cushion, now more sensitive to reductions in other gene levels
made genes “artificially” haploinsufficient
R7 precursor cell
signal from
cone photo- R8 neighbor
cell recptr
now other genes
in pathway
other genes in
ARE
pathway
NOT
haploinsufficient
haploinsufficient
sevenless/”+“
sevenless/+ (wildtype)
vs. sev/sev R7 photoreceptor
missing
(turned into cone cell)
adjust level to poise system
on phenotypic threshold
….then look for newly induced
dominant enhancer or suppressor alleles
Point to keep in mind:
…will not necessarily identify every relevant gene in pathway
this way
sevenless: receptor in R7 cell that responds to signal from R8
bride-of-sevenless: ligand (signal molecule) generated in R8
no new mutant alleles found
in sev sensitized screen!
Another way around the limitations of pleiotropy in genetic screens:
(2) use targetted genetic mosaics to screen for recessives
in the F1 (homozygous clones in heterozygotes
…in non-essential tissues only!)
…recover new recessives in the F1???
Based on a phenominon discovered (‘30s) by former chair
of U.C. Zoology Dept: mitotic recombination
but improved upon enormously in modern times
…only possible because of a very strange aspect of
fly chromosome behavior:
homologous chromosomes pair during mitotic interphase